Use of angiogenin or angiogenin agonists for treating diseases and disorders

ABSTRACT

The invention provides a method of treating a disorder characterised by elevated or dysregulated myostatin, disorders where the interaction between follistatin and angiogenin can be used to improve function in tissues, neurological diseases or disorders, spinal injuries or diseases, bone diseases or disorders, diseases involving glucose homeostasis, wound healing, neuro-protection, nervous system functional support or managing metabolic diseases, the method comprising administering an effective amount of angiogenin or an angiogenin agonist. Compositions and neutraceuticals comprising angiogenin are also provided.

FIELD

The present invention relates to methods for treating muscle disorders,including muscle wasting disorders and methods for improving muscle formby improving muscle function, strength, mass or exercise tolerance. Theinvention also relates to methods of decreasing fat, improving muscle tofat ratio and treating diseases caused by or involving suboptimal muscleto fat ratio. The invention also relates to treating diseases which canbe treated by improving follistatin mediated stimulation of cells.

BACKGROUND

Although muscle has its own progenitor cell for regeneration, lostmuscle bulk and strength due to disease and injury are often nevercompletely recovered. Therefore, treatments that can stimulate musclegrowth and prevent muscle loss are likely to benefit a significantproportion of the population.

Increase in muscle growth, weight or function is important for treatmentof deleterious conditions of the muscle, including, for example, muscledamage, muscle wasting, muscle degeneration, muscle atrophy or reducedrates of muscle repair. Such deleterious conditions of the muscle canresult from normal conditions of use or trauma, or quite frequently,through chronic disease states.

In addition to the various muscle disorders that may require treatment,improving muscle to fat ratio so as to have a greater lean mass has beenproposed to improve bone density. A correlation between lean mass andhigher total body bone density has been shown in mice and in men.Conversely people with higher fat mass have been shown to have reducedbone density. Accordingly improving muscle to fat ratio may improve bonedensity and be particularly useful in treating bone disorders such asosteoporosis.

Additionally perfectly healthy people may be desirous of improved muscleform or function. It may be desirous to improve a person's weightcarrying capacity, endurance, speed, or overall physique, all of whichcan be achieved by improving muscle mass or function. Additionally, itmay be desirous to improve the recovery of muscle from injury or reducethe time a muscle needs to recovery from extended use, for example toreduce the time between training for athletes, thereby improvingexercise tolerance.

In animal husbandry, such as involving animals as a food source, methodsthat increase the proportion and weight of muscle will greatly benefitthe industry.

Given the importance of this field a great deal of research is ongoingto develop methods of controlling muscle development or growth. Muchwork has centred on finding inhibitors of myostatin, as mysotatin, inadults, is a negative regulator of muscle growth (i.e. it suppressesmuscle growth).

Follistatin is a 35 kD glycoprotein that is synthesized in many tissuesand acts as a binding protein for activin and other members of the TGFβsuperfamily such as myostatin and some bone morphogenetic proteins.Follistatin is said to be one of several natural myostatin inhibitors,although its physiological role in muscle regulation is currentlyunknown. Nevertheless, administration of follistatin in muscle has beenobserved to lead to increased muscle mass, which is believed to be dueto its binding and neutralization of myostatin. One of the difficultiesof using follistatin as a therapeutic for increasing muscle growth isthat follistatin binds other TGFβ ligands besides myostatin, forexample, activin. Loss of activin activity in mice leads to numerousdevelopmental defects and neonatal death. Activin also limits growth ofmany types of epithelial tissue, so that inhibition of activin actionthrough administration of follistatin could lead to abnormal growth ofthese tissues and, eventually, to cancer.

There are currently no approved commercial pharmaceutical means forinhibiting myostatin activity that do not simultaneously alter activinactivity. Myostatin antibodies have been developed which bind andneutralize myostatin without binding other TGFβ family ligands. However,antibodies may have certain drawbacks that might limit their utility astherapeutics for muscle wasting disorders and certainly the use ofantibodies for muscle growth outside the therapeutic arena would be toocostly to be commercially useful.

It is an aim of a preferred embodiment of the present invention toaddress one or more of the above issues and ideally provide a treatmentfor muscle disorders for improving muscle function, strength, weightand/or exercise tolerance.

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. It will beclearly understood that, although a number of prior art publications arereferred to herein, this reference does not constitute an admission thatany of these documents forms part of the common general knowledge in theart.

SUMMARY

The invention generally provides methods of increasing muscle andreducing fat by administering angiogenin.

A first aspect provides a method of treating a disorder characterised byelevated or dysregulated myostatin in an individual, the methodcomprising administering an effective amount of angiogenin or anangiogenin agonist.

A second aspect provides a method of treating disorders where theinteraction between follistatin and angiogenin can be used to improvefunction in tissues by administering an effective amount of angiogeninor an angiogenin agonist.

A third aspect provides a method of promoting muscle growth in anindividual, the method comprising administering an effective amount ofangiogenin or an angiogenin agonist.

A fourth aspect provides a method of improving recovery of muscle frominjury or use in an individual, the method comprising administering aneffective amount of angiogenin or an angiogenin agonist.

A fifth aspect provides a method of improving muscle strength in anindividual, the method comprising administering an effective amount ofangiogenin or an angiogenin agonist.

A sixth aspect provides a method of improving exercise tolerance in anindividual, the method comprising administering an effective amount ofangiogenin or an angiogenin agonist.

A seventh aspect provides a method of increasing the proportion ofmuscle in an individual, the method comprising administering aneffective amount of angiogenin or an angiogenin agonist.

An eighth aspect provides a method of decreasing fat in an individual,the method comprising administering an effective amount of angiogenin oran angiogenin agonist.

A ninth aspect provides a method of decreasing an individual's fat tomuscle ratio, the method comprising administering an effective amount ofangiogenin or an angiogenin agonist.

Because of the link between muscle mass or muscle to fat ratio andinsulin sensitivity/metabolic syndrome (Guo T, Jou W, Chanturiya T,Portas J, Gavrilova O, McPherron A C. PLoS ONE. 2009; 4(3):e4937. Epub2009 Mar. 19), it is proposed that the methods of the seventh to ninthaspects may treat metabolic syndrome or enhance insulin sensitivity.

A tenth aspect provides a method for improving the bone density of anindividual by improving their muscle to fat ratio according to themethod of the ninth aspect.

It is proposed that angiogenin is capable of suppressing or reversingthe effect of myostatin as a negative regulator of muscle growth.

It is also proposed that myostatin and/or follistatin and/or angiogeninact on cells other than muscle cells; they may act on nerve cells, bonecells (oseoclasts) and endothelial cells.

Accordingly an eleventh aspect provides a method of treatingneurological diseases or disorders, spinal injuries or diseases, bonediseases or disorders, diseases involving glucose homeostasis, woundhealing, or for providing neuroprotection, nervous system functionalsupport and managing metabolic diseases, the method comprisingadministering an effective amount of angiogenin or an angiogeninagonist.

Whilst it is proposed that administration of angiogenin may act togetherwith endogenous follistatin, angiogenin or an angiogenin agonistadministered with follistatin (either simultaneously or sequentially)was shown by the inventors to have a more than additive effect comparedto administration of follistatin alone or angiogenin alone.

It will be appreciated that the converse of the inventors' findings willbe true, in that inhibitors or antagonists of angiogenin may be usefulfor treating diseases or conditions in which a reduction in musclegrowth or mass or an increase in fat or fat to muscle ratio or increasedmyostatin is desired.

The inventors were studying the effect of bovine angiogenin extractedfrom milk on human cells. They determined that bovine angiogenin iscapable of inducing vascular development of human umbilical veinendothelial cells (HUVEC) on matrigel in the same manner as humanvascular endothelial growth factor (VEGF).

The inventors then tested the effect of bovine angiogenin extracted frommilk in normal mice. The test group exhibited increased quadricep muscleweight and reduced abdominal fat pad weight when fed a diet includingbovine angiogenin. The demonstrated role of angiogenin in increasinglean muscle mass and decreasing fat mass indicates that methodsinvolving administering angiogenin or an angiogenin agonist have a broadvariety of applications where an increase in muscle tissue would betherapeutically beneficial, such as in livestock production, muscledisorders and for general fitness and physique. The invention may alsobe useful for treating diseases and disorders related to metabolism andadipose tissue.

The inventors finding is particularly surprising given the teaching ofthe prior art to administer follistatin to increase muscle mass andreduce fat mass. Without wishing to be bound by theory the inventorspropose that angiogenin and follistatin stimulate myotube formation andthat myostatin significantly inhibits myotube formation. Angiogenin isproposed to substantially reverse the effect of myostatin. The inventorspropose that the interaction between angiogenin and follistatin is amechanism that is of importance for stimulation, proliferation anddevelopment of cell types other than muscle alone. Therefore,administration of angiogenin can be used to treat conditions whereimproving follistatin mediated effects on cells is beneficial totreatment of disease or condition.

The suggestion that mechanism of action of angiogenin on muscle growthand fat loss is via its interaction with follistatin is supported by theinventors' in vitro studies, where treatment of muscle myoblasts witheither angiogenin or follistatin does not stimulate muscle growth overcontrol, whereas administration of both angiogenin and follistatin does.

In one embodiment of any one of the first to eleventh aspects angiogeninor angiogenin agonist is administered with follistatin.

In one embodiment of any one of the first to eleventh aspects angiogeninor angiogenin agonist is administered orally.

In one embodiment of any one of the first to eleventh aspects angiogeninor angiogenin agonist is administered orally and follistatin isadministered parentally.

A twelfth aspect provides a composition comprising angiogenin or anangiogenin agonist and follistatin.

In an embodiment of any of the first to twelfth aspects the angiogeninis recombinant angiogenin, preferably human or bovine recombinantangiogenin.

In an embodiment of any of the first to twelfth aspects the angiogeninis provided as an enriched extract from milk or plasma, particularlyfrom bovine milk or from bovine or human plasma. Such an enrichedextract is an angiogenin agonist, in that it is not pure angiogenin butprovides angiogenin activity.

Follistatin used in the methods or composition may be recombinant orprovided as an enriched extract from milk or plasma, particularly frombovine milk or from bovine or human plasma.

A thirteenth aspect provides a composition, food supplement orneutraceutical comprising angiogenin or an angiogenin agonist fortreating a disorder characterised by elevated myostatin, for treatingdisorders where the interaction between follistatin and angiogenin canbe used to improve function in tissues, for promoting muscle growth, forimproving recovery of muscle from injury or use, for improving musclestrength, for improving exercise tolerance, for increasing theproportion of muscle, for decreasing fat, for decreasing an individual'sfat to muscle ratio, for treating neurological diseases or disorders,for treating spinal injuries or diseases, for treating bone diseases ordisorders, for treating diseases involving glucose homeostasis, forwound healing, or for providing neuroprotection, nervous systemfunctional support, managing metabolic diseases and/or increasing thebone density of an individual.

A fourteenth aspect provides use of angiogenin or an angiogenin agonistin the manufacture of a medicament for treating a disorder characterisedby elevated myostatin, for treating disorders where the interactionbetween follistatin and angiogenin can be used to improve function intissues, for promoting muscle growth, for improving recovery of musclefrom injury or use, for improving muscle strength, for improvingexercise tolerance, for increasing the proportion of muscle, fordecreasing fat, for decreasing an individual's fat to muscle ratio,and/or increasing the bone density of an individual.

In an embodiment of the fourteenth aspect the medicament also comprisesfollistatin.

In another embodiment of the fourteenth aspect the medicament is foradministering to an individual being treated with follistatin.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows human endothelial cells (HUVEC) photographed at 10×magnification. A shows vascular development caused by treatment withangiogenin (100 ng/ml), B shows positive control VEGF (10 ng/ml) and Cis the negative control.

FIG. 2 shows a bar graph illustrating growth of murine C2C12 myoblastsin vitro when administered casein, BSA, follistatin, angiogenin, orfollistatin+angiogenin or a positive control (DMEM and 10% FCS).

FIG. 3 shows bAngiogenin and hAngiogenin can induce myoblastdifferentiation into myotubes in the absence of serum in a dosedependant manner. C2C12 myoblast cells were cultured in 6 well plates inDMEM (control) supplemented with bAngiogenin (bANG) or hAngiogenin(hANG). The images taken after 96 hours show that both bANG and hANGinduce myotube formation compared to the control DMEM culture.

FIG. 4 shown bovine Angiogenin induces myotube formation in the presenceof 2% HS in a dose dependant manner. C2C12 myoblast cells were culturedin differentiation media (DMEM+2% HS; control) supplemented withbAngiogenin (bANG) or hAngiogenin (hANG). The images taken after 96hours show that bANG induces myotube formation compared to the controlculture. rhANG proves that angiogenin is the inducing factor

FIG. 5 shows bovine Angiogenin interacts with FS to enhance myotubeformation. C2C12 myoblast cells were cultured in differentiation media(DMEM+2% HS; control) supplemented with bovine Angiogenin (bANG),Follistatin (FS) or combined. The images (a) and CK analysis (b) at 96hours show that bANG interacts with FS to induces myotube formationsynergistically compared to the either reagent in isolation.

FIG. 6 shows hierarchical clustering of differentially expressed genes(based on a fold change of at least ±1.6 and P<0.05) in C2C12 cellsafter 2 hrs of differentiation to form myotubes. C2C12 myoblast cellswere cultured in differentiation media (DMEM+2% HS; control)supplemented with bovine Angiogenin, Follistatin or combined Genesshowing increased expression are represented in red, genes withdecreased expression are represented in blue, genes with no change inexpression are represented in yellow.

FIG. 7 shows Angiogenin Blocking Peptide inhibits myotube formation. Thepeptide (VFSVRVSILVF) specifically blocks the angiogenin/actininteraction and inhibits the angiogenin induction of myotube formation.

FIG. 8 shows bovine Angiogenin can regulate myostatin effects on myotubeformation. Angiogenin is able to negate the negative effect of myostatin(Myo) on muscle myotube formation. The Angiogenin-Follistatinsynergistic mechanism recovers tube formation to control levels in thepresence of myostatin.

FIG. 9 shows angiogenin can regulate myostatin effects on myotubeformation. Angiogenin is able to negate the negative effect of myostatin(Myo) on muscle myotube formation. The Angiogenin-Follistatinsynergistic mechanism recovers tube formation to control levels in thepresence of myostatin.

FIGS. 10 and 11 show protection of PC12 cells against cell death uponserum starvation in the presence of rhAngiogenin+rhFollistatin relativeto rhAngiogenin alone or rhFollistatin alone. Results are presented asMean+SEM of replicate cultures (12 replicates for medium only control; 6replicates for rhAngiogenin (1.0 μg/ml), bAngiogenin (10 μg/ml) andrhFollistatin (0.1 μg/ml); 3 replicates for rhNGF controls).

FIG. 12 shows angiogenin fed in the diet at 2.5 μg/g feed under adlibitum feeding conditions increases quadriceps weight in mice fed for 1month and allowed to exercise freely on standard rodent running wheels.

FIG. 13 shows that angiogenin fed in the diet at 2.5 μg/g feed under adlibitum feeding conditions increases results in muscle fibre type crosssectional area (SA) changes in mice fed for 1 month and allowed toexercise freely on standard rodent running wheels. Group means forcontrol animals are represented in white bars and group means forangiogenin treated animals are represented in black bars. Standarddeviations are given.

FIG. 14 shows that angiogenin fed in the diet at 2.5 μg/g feed under adlibitum feeding conditions increases distance run per day in mice fedfor 1 month and allowed to exercise freely on standard rodent runningwheels.

FIG. 15 shows that that angiogenin fed in the diet at 2.5 μg/g feedunder ad libitum feeding conditions reduces the area of muscle necrosisin the quadriceps of MDX mice allowed to exercise freely on standardrodent running wheels.

DETAILED DESCRIPTION

Angiogenin is a 14 kDa, non-glycosylated polypeptide which is producedby several growing cell types including vascular endothelial cells,aortic smooth muscle cells, fibroblasts, and some tumours such as coloncarcinomas, ovarian carcinomas, and breast cancers. Angiogenin has beenisolated from a number of sources including normal human plasma, bovineplasma, bovine milk, and mouse, rabbit and pig sera.

Angiogenin is homologous to pancreatic ribonuclease and has distinctribonucleolytic activity. The protein is able to induce new blood vesselgrowth; however, it has not been established what role theribonucleolytic activity of angiogenin plays in angiogenesis induced bythis protein.

As well as a potent stimulator of angiogenesis, angiogenin has beenshown to possess a number of other activities. However there is noprevious disclosure of angiogenin's effect on muscle other than viaincreasing angiogenesis.

The inventors have shown that angiogenin rich purifications derived bycation-exchange chromatography of milk fractions also containfollistatin, a protein of significantly different charge properties toangiogenin (data not shown). In the prior art, angiogenin andfollistatin have been shown to bind to each other in a yeast two-hybridmodel. The inventors show for the first time a biologically significantinteraction between angiogenin and follistatin in mammalian cells.Follistatin is known as an antagonist of myostatin, a protein said tocontrol muscle growth and development.

The invention in one aspect relates to the treatment of disorders. Theterms “treating” and “treatment” as used herein refer to reduction inseverity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms (prophylaxis)and/or their underlying cause, and improvement or remediation of damage.Thus, for example, the present method of “treating” a disorderencompasses both prevention of the disorder in a predisposed individualand treatment of the disorder in a clinically symptomatic individual.

“Treating” as used herein covers any treatment of, or prevention of acondition in a vertebrate, a mammal, particularly a human, and includes:inhibiting the condition, i.e., arresting its development; or relievingor ameliorating the effects of the condition, i.e., cause regression ofthe effects of the condition.

“Prophylaxis” or “prophylactic” or “preventative” therapy as used hereinincludes preventing the condition from occurring or ameliorating thesubsequent progression of the condition in a subject that may bepredisposed to the condition, but has not yet been diagnosed as havingit.

In the prior art myostatin is said to play a role in muscle developmentand a number of related disorders or diseases. In adults, myostatin mRNAis primarily detected in skeletal muscle although lower concentrationsare also found in adipose tissue and cardiac tissue. Myostatin knockoutmice have two- to three-fold greater muscle mass than their wild typelittermates. The increased muscle mass is the result of fibrehypertrophy and hyperplasia. In addition, the myostatin knockout miceaccumulate less fat than their wild type littermates but otherwiseappear normal and healthy. Myostatin has also been recently shown to bean important regulator of adipogenesis. Additionally, bone structure andcontent has been recently studied in myostatin deficient mice.

Since the inventors propose that myostatin actually antagonises theeffect of angiogenin on the muscle, they propose that angiogenin can beused to treat any disease in which inhibition of myostatin haspreviously been suggested.

Accordingly angiogenin may be used in accordance with the presentinvention to increase muscle mass, increase bone density, decreasemuscle wasting, or may be useful for the treatment or prevention ofconditions wherein the presence of myostatin causes or contributes toundesirable pathological effects or decrease of myostatin levels has atherapeutic benefit in mammals, preferably humans. In addition,angiogenin may be used to treat conditions where myostatin is notdysregulated, but improved follistatin mediated cell stimulation can begained by addition of exogenous angiogenin.

Angiogenin can be used to reduce the severity of a pathologic condition,which is characterized, at least in part, by an abnormal amount,development or metabolic activity of muscle or adipose tissue in asubject. It can be administered to prevent, ameliorate or reduce theseverity of a wasting disorder, such as cachexia, anorexia, AIDS wastingsyndrome, muscular dystrophies, neuromuscular diseases, motor neurondiseases, diseases of the neuromuscular junction, and inflammatorymyopathies.

The term “disorder associated with myostatin” refers to disorders ofmuscle, bone, or glucose homeostasis, and include disorders associatedwith abnormal myostatin.

The invention extends to treatment of muscular disorders and of diseasesassociated with muscular degeneration characteristics. Non limitingexamples of such disorders are various neuromuscular diseases, cardiacinsufficiency, weakness of single muscles such as e.g. the constrictoror bladder muscle, hypo- or hypertension caused by problems with theconstrictor function of vascular smooth muscle cells, impotence/erectiledysfunction, incontinence, AIDS-related muscular weakness, and generaland age-related amyotrophia.

Disorders of muscle as referred to herein particularly include musclewasting conditions or disorders in which muscle wasting is one of theprimary symptoms.

A muscle is a tissue of the body that primarily functions as a source ofpower. There are three types of muscles in the body: a) skeletalmuscle—striated muscle responsible for generating force that istransferred to the skeleton to enable movement, maintenance of postureand breathing; b) cardiac muscle—the heart muscle; and c) smoothmuscle—the muscle that is in the walls of arteries and bowel. Themethods of the invention are particularly applicable to skeletal musclebut may have some effect on cardiac and or smooth muscle.

Skeletal muscle fibers are generally classified as type I(oxidative/slow) or type II (glycolytic/fast) fibers. They displaymarked differences in respect to concentration, metabolism, andsusceptibility to fatigue. Type I fibers are mitochondria-rich andmainly use oxidative metabolism for energy production, which provides astable and long-lasting supply of ATP, and thus are fatigue-resistant.Type II fibers comprise three sub-types: IIa, IIx, and IIb. Type IIbfibers have the lowest levels of mitochondrial content and oxidativeenzymes, rely on glycolytic metabolism as major energy source, and aresusceptible to fatigue, while the oxidative and contraction functions oftype IIa and IIx lie between type I and IIb. Adult skeletal muscle showsplasticity and can undergo conversion between different fiber types inresponse to exercise training or modulation of motoneuron activity.

Determination of the muscle fiber composition in athletes revealed thatelite endurance athletes have relatively more type I fibers than type IIfibers in the trained musculature. Marathon runners also tend to havemore type I fibers. It was suggested that type I fiber might be a factorgoverning physical endurance capacity.

On the contrary, ageing and physical inactivity are conditionsassociated with a decrease in type I fibers, oxidative capacity andinsulin sensitivity. It appears that the muscle oxidative capacity is acrucial factor for determining endurance and fatigue resistance. Thereseem to be an adaptive metabolic response of skeletal muscle toendurance exercise by controlling the number of oxidative muscle fibers(type I fibers).

The conversion of skeletal muscle fiber type IIb to type IIa and type Iis regulated by different signalling pathways. For example theRas/mitogen-activated protein kinase (MAPK), calcineurin,calcium/calmodulin-dependent protein kinase FV and the peroxisomeproliferator y coactivator 1 (PGC-I). Angiogenin may modulate thesepathways and such may have an influence on the skeletal muscle fibers.

“Muscle wasting” refers to the progressive loss of muscle mass and/or tothe progressive weakening and degeneration of muscles, including theskeletal or voluntary muscles which control movement, cardiac muscleswhich control the heart, and smooth muscles. In one embodiment, themuscle wasting condition or disorder is a chronic muscle wastingcondition or disorder. “Chronic muscle wasting” is defined herein as thechronic (i.e. persisting over a long period of time) progressive loss ofmuscle mass and/or to the chronic progressive weakening and degenerationof muscle.

The loss of muscle mass that occurs during muscle wasting can becharacterized by a muscle protein breakdown or degradation, by muscleprotein catabolism. Protein catabolism occurs because of an unusuallyhigh rate of protein degradation, an unusually low rate of proteinsynthesis, or a combination of both. Protein catabolism or depletion,whether caused by a high degree of protein degradation or a low degreeof protein synthesis, leads to a decrease in muscle mass and to musclewasting. The term “catabolism” has its commonly known meaning in theart, specifically an energy burning form of metabolism.

Muscle wasting can occur as a result of a pathology, disease, conditionor disorder. In one embodiment, the pathology, illness, disease orcondition is chronic. In another embodiment, the pathology, illness,disease or condition is genetic. In another embodiment, the pathology,illness, disease or condition is neurological. In another embodiment,the pathology, illness, disease or condition is infectious. As describedherein, the pathologies, diseases, conditions or disorders for which thecompounds and compositions of the present invention are administered arethose that directly or indirectly produce a wasting (i.e. loss) ofmuscle mass, that is a muscle wasting disorder.

Especially preferred is the treatment of neuromuscular diseases whichare aligned with joint or skeletal deformities. In one embodiment,muscle wasting in a subject is a result of the subject having a musculardystrophy; muscle atrophy; or X-linked spinal-bulbar muscular atrophy(SBMA).

The muscular dystrophies are genetic diseases characterized byprogressive weakness and degeneration of the skeletal or voluntarymuscles that control movement. The muscles of the heart and some otherinvoluntary muscles are also affected in some forms of musculardystrophy. The major forms of muscular dystrophy (MD) are: duchennemuscular dystrophy, myotonic dystrophy, becker muscular dystrophy,limb-girdle muscular dystrophy, facioscapulhumeral muscular dystrophy,congenital muscular dystrophy, oculopharyngeal muscular dystrophy,distal muscular dystrophy and emery-dreifuss muscular dystrophy.

Muscular dystrophy can affect people of all ages. Although some formsfirst become apparent in infancy or childhood, others may not appearuntil middle age or later. Duchenne MD is the most common form,typically affecting children. Myotonic dystrophy is the most, common ofthese diseases in adults.

Muscle atrophy (MA) is characterized by wasting away or diminution ofmuscle and a decrease in muscle mass. For example, Post-Polio MA is amuscle wasting that occurs as part of the post-polio syndrome (PPS). Theatrophy includes weakness, muscle fatigue, and pain.

Another type of MA is X-linked spinal-bulbar muscular atrophy (SBMA—alsoknown as Kennedy's Disease). This disease arises from a defect in theandrogen receptor gene on the X chromosome, affects only males, and itsonset is in adulthood.

Sarcopenia is a debilitating disease that afflicts the elderly andchronically ill patients and is characterized by loss of muscle mass andfunction. Further, increased lean body mass is associated with decreasedmorbidity and mortality for certain muscle-wasting disorders. Inaddition, other circumstances and conditions are linked to, and cancause muscle wasting disorders. For example, studies have shown that insevere cases of chronic lower back pain, there is paraspinal musclewasting.

Muscle wasting and other tissue wasting is also associated with advancedage. It is believed that general weakness in old age is due to musclewasting. As the body ages, an increasing proportion of skeletal muscleis replaced by fibrous tissue. The result is a significant reduction inmuscle power, performance and endurance.

Long term hospitalization due to illness or injury, or disusedeconditioning that occurs, for example, when a limb is immobilized, canalso lead to muscle wasting, or wasting of other tissue. Studies haveshown that in patients suffering injuries, chronic illnesses, burns,trauma or cancer, who are hospitalized for long periods of time, thereis a long-lasting unilateral muscle wasting, and a decrease in bodymass.

Injuries or damage to the central nervous system (CNS) are alsoassociated with muscle wasting and other wasting disorders. Injuries ordamage to the CNS can be, for example, caused by diseases, trauma orchemicals. Examples are central nerve injury or damage, peripheral nerveinjury or damage and spinal cord injury or damage. In one embodiment CNSdamage or injury comprise Alzheimer's diseases (AD); stroke, anger(mood); anorexia, anorexia nervosa, anorexia associated with agingand/or assertiveness (mood).

In another embodiment, muscle wasting or other tissue wasting may be aresult of alcoholism.

In one embodiment, the wasting disease, disorder or condition beingtreated is associated with chronic illness

This embodiment is directed to treating, in some embodiments, anywasting disorder, which may be reflected in muscle wasting, weight loss,malnutrition, starvation, or any wasting or loss of functioning due to aloss of tissue mass.

In some embodiments, wasting diseases or disorders, such as cachexia;malnutrition, tuberculosis, leprosy, diabetes, renal disease, chronicobstructive pulmonary disease (COPD), cancer, end stage renal failure,sarcopenia, emphysema, osteomalacia, or cardiomyopathy, may be treatedby the methods of this invention

In some embodiments, wasting is due to infection with enterovirus,Epstein-Barr virus, herpes zoster, HIV, trypanosomes, influenze,coxsackie, rickettsia, trichinella, schistosoma or mycobacteria.

Cachexia is weakness and a loss of weight caused by a disease or as aside effect of illness. Cardiac cachexia, i.e. a muscle protein wastingof both the cardiac and skeletal muscle, is a characteristic ofcongestive heart failure. Cancer cachexia is a syndrome that occurs inpatients with solid tumors and hematological malignancies and ismanifested by weight loss with massive depletion of both adipose tissueand lean muscle mass.

Cachexia is also seen in acquired immunodeficiency syndrome (AIDS),human immunodeficiency virus (HIV)-associated myopathy and/or muscleweakness/wasting is a relatively common clinical manifestation of AIDS.Individuals with HIV-associated myopathy or muscle weakness or wastingtypically experience significant weight loss, generalized or proximalmuscle weakness, tenderness, and muscle atrophy.

Untreated muscle wasting disorders can have serious health consequences.The changes that occur during muscle wasting can lead to a weakenedphysical state resulting in poor performance of the body and detrimentalhealth effects.

Thus, muscle atrophy can seriously limit the rehabilitation of patientsafter immobilizations. Muscle wasting due to chronic diseases can leadto premature loss of mobility and increase the risk of disease-relatedmorbidity. Muscle wasting due to disuse is an especially serious problemin elderly, who may already suffer from age-related deficits in musclefunction and mass, leading to permanent disability and premature deathas well as increased bone fracture rate. Despite the clinical importanceof the condition few treatments exist to prevent or reverse thecondition. The inventors propose that angiogenin can be used to preventand treat muscle wasting or atrophy associated with any of theconditions recited above.

Angiogenin, particularly in combination with follistatin or whenadministered orally is shown herein to be neuroprotective and hence findutility in treating neurological disorders or diseases affecting thenervous system, particularly motor neurone diseases. Exemplary motorneuron diseases that can be treated with angiogenin include AmyotrophicLateral Sclerosis (ALS) (also known as Lou Gehrig's Disease), InfantileProgressive Spinal Muscular Atrophy (SMA, SMAl or WH) (also known as SMAType 1, Werdnig-Hoffman), Intermediate Spinal Muscular Atrophy (SMA orSMA2) (also known as SMA Type 2), Juvenile Spinal Muscular Atrophy (SMA,SMA3 or KW) (also known as SMA Type 3, Kugelberg-Welander), SpinalBulbar Muscular Atrophy (SBMA) (also known as Kennedy's Disease andX-Linked SBMA), and Adult Spinal Muscular Atrophy (SMA).

Exemplary inflammatory myopathies that can be treated with angiogenininclude Dermatomyositis (PM/DM), Polymyositis (PM/DM), and InclusionBody Myositis (IBM).

Exemplary diseases of the neuromuscular junction that can be treatedwith angiogenin include: Myasthenia Gravis (MG), Lambert-Eaton Syndrome(LES), and Congenital Myasthenic Syndrome (CMS).

Exemplary myopathies due to endocrine abnormalities that can be treatedwith angiogenin include Hyperthyroid Myopathy (HYPTM) and HypothyroidMyopathy (HYPOTM).

Exemplary diseases of peripheral nerve that can be treated withangiogenin include Charcot-Marie-Tooth Disease (CMT), Dejerine-SottasDisease (DS), and Friedreich's Ataxia (FA).

Other exemplary myopathies that can be treated with angiogenin includeMyotonia Congenita (MC), Paramyotonia Congenita (PC), Central CoreDisease (CCD), Nemaline Myopathy (NM), Myotubular Myopathy (MTM or MM),and Periodic Paralysis (PP).

Angiogenin can also be used to promote wound healing and to treatwounds, both of which uses have previously been proposed for myostatininhibitors.

Exemplary metabolic diseases of muscle that can be treated withangiogenin include Phosphorylase Deficiency (MPD or PYGM), Acid MaltaseDeficiency (AMD), Phosphofructokinase Deficiency (PFKM), DebrancherEnzyme Deficiency (DBD), Mitochondrial Myopathy (MITO), CarnitineDeficiency (CD), Carnitine Palmityl Transferase Deficiency (CPT),Phosphoglycerate Kinase Deficiency (PGK), Phosphoglycerate MutaseDeficiency (PGAM or PGAMM), Lactate Dehydrogenase Deficiency (LDHA), andMyoadenylate Deaminase Deficiency (MAD). These diseases have previouslybeen proposed to be treated by myostatin inhibitors.

In the accompanying experiments the inventors show that angiogeninreduces fat. This has previously been shown for myostatin inhibitors andshows that angiogenin can be used to treat disease connected to impairedlipid metabolism such as dyslipidemia and related lipid abnormalitiessuch as hyperlipidemia, hypercholesteremia, hypertriglyceridemia andmixed dyslipidemia.

Dyslipidemia is characterized by abnormalities in circulating lipidlevels due to alterations in lipid metabolism. These abnormalities caninclude any one or several of the different circulating lipid fractions(cholesterol, triglyceride, lipoprotein). Dyslipidemia includeshypercholesterolemia, which is an elevation of serum cholesterol abovethe normal limit (normal safe limit is approximately in the range of125-200 mg/dl in human blood), hypertriglyceridemia which is an increaseof serum triglycerides above the normal level (normal safe limit isapproximately in the range of 30-140 mg/dl in human blood) and mixedlipid disorders.

Dyslipidemia includes hypertriglyceridemia and mixed dyslipidemia(hyperlipidemia). Hypertriglyceridemia involves a rise in the levels ofvery low density lipoprotein (VLDL), while mixed dyslipidemia(hyperlipidemia) involves a combination both hypertriglyceridemia andhypercholesterolemia and is also often associated with a drop in highdensity lipoprotein (HDL) levels. Thus, dyslipidemia is also a disorderof lipoprotein metabolism that results in an overproduction or adeficiency of lipoproteins. Dyslipidemia is typically characterized byany one or more of the following: elevated plasma triglycerides,elevated total plasma cholesterol, low High Density Lipoproteincholesterol (HDL-c), elevated levels of Low Density Lipoproteincholesterol (LDL-c). For example, dyslipidemia may be one or more of thefollowing conditions: low HDL-c (<35 or 40 mg/dl), high triglycerides(>200 mg/dl), high LDL-c (>150 mg/dl), elevated cholesterol (>200mg/dl).

Dyslipidemia is widely considered as one of the main risk factor forcardiovascular vascular diseases (CVD) and atherogenesis. Cardiovasculardisorders are among the leading causes of disability and deathworldwide. High serum cholesterol, particularly cholesterol associatedwith LDL and VLDL, is one of the principal risk factors foratherogenesis. High triglycerides, increased small LDL, and decreasedHDL levels all appear to be independently atherogenic. There is a stronginverse association between plasma HDL and the risk of CVD. A positiveassociation exists between LDL cholesterol and risk of CVD. Thus, therisk of coronary artery disease increases when LDL and VLDL levelsincrease while high levels of cholesterol carried in HDL is protectiveagainst coronary artery disease. Triglycerides also seem to play animportant role in CVD. High level of fasting triglycerides is a strongrisk factor for ischaemic heart disease in elderly men independently ofother major risk factors including HDL-cholesterol. People with combinedhyperlipidemia, which is characterized by elevated serum levels of bothcholesterol and triglycerides, run a higher risk of heart disease thanthose with only a high LDL cholesterol level. Therefore, lowering bothlevels is a desired goal.

Diseases connected to impaired glucose metabolism and impaired insulinaction include diabetes mellitus, especially diabetes mellitus type 1and 2, more especially (non-autoimmune) non-insulin dependent diabetesmellitus (NIDDM; so called Type 2 Diabetes). Another such disease issyndrome X or metabolic syndrome.

Diabetes mellitus defines a complex of metabolic diseases derived frommultiple causative factors and is characterized by impaired glucosemetabolism, usually associated with impaired protein and fat metabolism.This results in elevated fasting and postprandial serum glucose thatleads to complications if left untreated. Four different forms ofdiabetes mellitus are known, (1) type 1 diabetes mellitus, (2) type 2diabetes mellitus, (3) the so-called gestational diabetes mellitus,which begins or is recognized for the first time during pregnancy, and(4) some other forms which are mainly based on genetic defects.

The term “diabetes mellitus” includes, but is not limited to, metabolicabnormalities such as increased blood glucose level, obesity associatedpathologies, impaired glucose tolerance, increased insulin resistance,hyperlipidemia, dyslipidemia, increase in cholesterol(hypercholesterinemia, hypertriglycerinemia), hyperinsulinemia,hypertension, and microalbuminuria. Impaired glucose tolerance andimpaired fasting glucose are the two symptoms referred to aspre-diabetes mellitus. This stage is associated with the so-calledinsulin resistance, one of a group of metabolic diseases called“syndrome X” or “metabolic syndrome”, particularly associated with ahigh fat to muscle ratio. Since type 2 diabetes mellitus is oftenassociated with other symptoms from syndrome X, such ashypertriglyceridemia or dyslipidemia, and the use of angiogenin shouldgreatly improve the fat to muscle ratio of a subject the methods of thepresent invention are also useful for the treatment or prevention ofsyndrome X.

The two major forms of diabetes mellitus are the type 1 and type 2diabetes mellitus, of which type 2 diabetes mellitus is the mostprevailing form. Type 1 and type 2 diabetes mellitus are associated withhyperglycemia, hypercholesterolemia and hyperlipidemia. Theinsensitivity to insulin and absolute insulin deficiency in type 1 and 2diabetes mellitus leads to a decrease in glucose utilization by theliver, muscle and the adipose tissue and to increased blood glucoselevels. Uncontrolled hyperglycemia is associated with the dysfunctionand failure of various organs such as the eyes, heart, blood vessels,kidney and nerves thus leading to increased and premature mortality dueto an increased risk for microvascular and macrovascular diseases,including nephropathy, neuropathy, retinopathy, ulceration of the legsand feet, fatty liver disease, hypertension, cardiovascular diseases,and cerebrovascular diseases (stroke), the so-called diabeticcomplications.

Recent evidence showed that tight glycemic control is a major factor inthe prevention of these complications in both type 1 and type 2 diabetesmellitus. Therefore, optimal glycemic control by drugs or therapeuticregimens is an important approach for the treatment of diabetesmellitus.

Type 1 diabetes mellitus is the form of diabetes mellitus which usuallybegins with childhood or puberty and is characterized by an auto-immunedestruction of the insulin-producing β-cells leading to a completedeficiency of insulin secretion.

Type 2 diabetes mellitus is the form of diabetes mellitus which occurspredominantly in adults in whom adequate production of insulin isavailable in the early stage of the diseases, yet a defect exists ininsulin sensitivity, especially in insulin-mediated utilization andmetabolism of glucose in peripheral tissues. The changes in varioustissues associated with type 2 diabetes mellitus exist even beforeclinical symptoms are detected.

Also contemplated is the treatment of insulin resistance induced bytrauma (e.g. burns or nitrogen imbalance) and adipose tissue disorders(e.g. obesity).

Other uses for angiogenin in accordance with the invention include fortreatment of osteoporosis, especially in the elderly and/orpostmenopausal women; glucocorticoid-induced osteoporosis; osteopenia;osteoarthritis; osteoporosis-related fractures; and traumatic or chronicinjury to muscle tissue. Further uses for angiogenin include treatmentof low bone mass due to chronic glucocorticoid therapy, prematuregonadal failure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

The invention in other aspects also contemplates treating healthyindividuals to cause an increase in muscle mass, strength, function oroverall physique. Angiogenin is also proposed to promote muscle recoveryfrom injury or trauma or damage or overuse through training andtherefore to increase exercise tolerance.

The term “increase in muscle mass” refers to the presence of a greateramount of muscle after treatment with angiogenin relative to the amountof muscle mass present before the treatment.

The term “increase in muscle strength” refers to the presence of amuscle with greater force generating capacity after treatment withangiogenin relative to that present before the treatment.

The term “increase in muscle function” refers to the presence of musclewith greater variety of function after treatment with angiogeninrelative to that present before the treatment.

The term “increase in exercise tolerance” refers to the ability toexercise with less rest between exercise after treatment with angiogeninrelative to that needed before the treatment.

A muscle is a tissue of the body that primarily functions as a source ofpower. There are three types of muscles in the body: a) skeletalmuscle—striated muscle responsible for generating force that istransferred to the skeleton to enable movement, maintenance of postureand breathing; b) cardiac muscle—the heart muscle; and c) smoothmuscle—the muscle that is in the walls of arteries and bowel. Themethods of the invention are particularly applicable to skeletal musclebut may have some effect on cardiac and or smooth muscle. Reference toskeletal muscle as used herein also includes interactions between bone,muscle and tendons and includes muscle fibres and joints.

Whilst angiogenin has previously been suggested to have an effect oncardiac muscle by virtue of its angiogenic activity and ability toprovide increased blood flow to a muscle, this effect was restricted tooxidative muscles (type I and type IIa). The follistatin mediatedeffects of angiogenin on muscle as seen in the present invention aredistinct from those relating to angiogenesis as evidenced by all musclefibres being affected.

The term “decrease in fat” refers to the presence of a reduced amount offat after treatment with angiogenin relative to the amount of fatpresent before the treatment. The present invention is particularlyapplicable to visceral fat, fat located inside the peritoneal cavity andaround internal organs. It may also effect subcutaneous fat and/orintramuscular fat.

The proposed uses of angiogenin on healthy individuals will be useful toathletes, both elite and amateur, body builders, those desirous ofweight loss of enhanced physique and manual workers.

Since angiogenin is highly conserved in sequence and function acrossspecies, the methods of the invention are applicable in non-humanmammals or avian species [e.g. domestic animals (e.g., canine andfeline), sports animals (e.g., equine), food-source animals (e.g.,bovine, porcine and ovine), avian species (e.g., chicken, turkey, othergame birds or poultry)] wherein the presence of myostatin causes orcontributes to undesirable pathological effects or decrease of myostatinlevels has a therapeutic benefit.

The angiogenin or angiogenin agonist may be provided as apharmaceutical, veterinary or neutraceutical composition or as a food.

A pharmaceutical composition is one which is suitable for administrationto humans. A veterinary composition is one that is suitable foradministration to animals. Generally such compositions will containpurified angiogenin or angiogenin agonist or at the very least allcomponents of the composition will be verifiable.

The compositions used in the methods of the first to eleventh aspectsmay comprise one or more carriers and optionally other therapeuticagents. Each carrier, diluent, adjuvant and/or excipient may bepharmaceutically “acceptable”.

By “pharmaceutically acceptable carrier” is meant a material which isnot biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected active agentwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. Similarly, a“pharmaceutically acceptable” salt or ester of a novel compound asprovided herein is a salt or ester which is not biologically orotherwise undesirable.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering the agentto the subject. The carrier may be liquid or solid and is selected withthe planned manner of administration in mind. Each carrier must bepharmaceutically “acceptable” in the sense of being not biologically orotherwise undesirable i.e. the carrier may be administered to a subjectalong with the agent without causing any or a substantial adversereaction.

The composition may be administered orally, topically, or parenterallyin formulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles.

The term parenteral as used herein includes intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, subconjunctival,intracavity, transdermal and subcutaneous injection, aerosol foradministration to lungs or nasal cavity or administration by infusionby, for example, osmotic pump.

The composition may be administered orally as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. The composition for oral use may contain one or moreagents selected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharin. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable preservatives includesodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methylparaben, propyl paraben or sodium bisulphite. Suitable lubricantsinclude magnesium stearate, stearic acid, sodium oleate, sodium chlorideor talc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate. The tablets may contain the agent in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets.

These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

The compositions may also contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions. Thepharmaceutical compositions may also be included in a container, pack,or dispenser together with instructions for administration.

Other therapeutically useful agents, such as growth factors (e.g., BMPs,TGF-P, FGF, IGF), cytokines (e.g., interleukins and CDFs), antibiotics,and any other therapeutic agent beneficial for the condition beingtreated may optionally be included in or administered simultaneously orsequentially with the angiogenin or angiogenin agonist.

Angiogenin or its agonists may also be presented for use in the form ofveterinary compositions, which may be prepared, for example, by methodsthat are conventional in the art. Examples of such veterinarycompositions include those adapted for:

(a) oral administration, external application, for example drenches(e.g. aqueous or non-aqueous solutions or suspensions); tablets orboluses; powders, granules or pellets for admixture with feed stuffs;pastes for application to the tongue, particularly adapted forprotection through the rumen if to be administered to ruminants;

(b) parenteral administration for example by subcutaneous, intramuscularor intravenous injection, e.g. as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied tothe skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

It is especially advantageous to formulate the compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Compositions comprising angiogenin or an agonist thereof are to beadministered in therapeutically effective amounts. As used herein, an“effective amount” of angiogenin is a dosage which is sufficient toreduce the activity of myostatin to achieve a desired biologicaloutcome. The desired biological outcome may be any therapeutic benefitincluding an increase in muscle mass, an increase in muscle strength,improved metabolism, decreased adiposity, or improved glucosehomeostasis. Such improvements may be measured by a variety of methodsincluding those that measure lean and fat body mass (such as duel rayscanning analysis), muscle strength, serum lipids, serum leptin, serumglucose, glycated hemoglobin, glucose tolerance, and improvement in thesecondary complications of diabetes.

Generally, a therapeutical effective amount may vary with the subject'sage, condition, and sex, as well as the severity of the medicalcondition in the subject. The dosage may be determined by an physicianand adjusted, as necessary, to suit observed effects of the treatment.Appropriate dosages for administering angiogenin or its agonists mayrange from 5 mg to 100 mg, from 15 mg to 85 mg, from 30 mg to 70 mg, orfrom 40 mg to 60 mg. The compositions can be administered in one dose,or at intervals such as once daily, once weekly, and once monthly.

Dosage schedules can be adjusted depending on the half life ofangiogenin or its agonist, or the severity of the patient's condition.

Generally, the compositions are administered as a bolus dose, tomaximize the circulating levels of angiogenin for the greatest length oftime after the dose. Continuous infusion may also be used after thebolus dose.

It is also contemplated that the methods utilise a neutraceuticalcomposition to provide the angiogenin. A neutraceutical composition foruse in the methods is provided.

The term “nutraceutical” as used herein refers to an edible productisolated or purified from food, in this case from a milk product, whichis demonstrated to have a physiological benefit or to provide protectionor attenuation of an acute or chronic disease or injury when orallyadministered. The nutraceutical may thus be presented in the form of adietary preparation or supplement, either alone or admixed with ediblefoods or drinks.

The nutraceutical composition may be in the form of a soluble powder, aliquid or a ready-to-drink formulation. Alternatively, the nutritionalcomposition may be in solid form as a food; for example in the form of aready-to-eat bar or breakfast cereal. Various flavours, fibres,sweeteners, and other additives may also be present.

The nutraceutical preferably has acceptable sensory properties (such asacceptable smell, taste and palatability), and may further comprisevitamins and/or minerals selected from at least one of vitamins A, B1,B2, B3, B5, B6, B11, B12, biotin, C, D, E, H and K and calcium,magnesium, potassium, zinc and iron.

The nutraceutical composition may be produced as is conventional; forexample, the composition may be prepared by blending together theprotein and other additives. If used, an emulsifier may be included inthe blend. Additional vitamins and minerals may be added at this pointbut are usually added later to avoid thermal degradation.

If it is desired to produce a powdered nutraceutical composition, theprotein may be admixed with additional components in powdered form. Thepowder should have a moisture content of less than about 5% by weight.Water, preferably water which has been subjected to reverse osmosis, maythen be mixed in to form a liquid mixture.

If the nutraceutical composition is to be provided in a ready to consumeliquid form, it may be heated in order to reduce the bacterial load. Ifit is desired to produce a liquid nutraceutical composition, the liquidmixture is preferably aseptically filled into suitable containers.Aseptic filling of the containers may be carried out using techniquescommonly available in the art. Suitable apparatus for carrying outaseptic filling of this nature is commercially available.

Preferably the neutraceutical composition also comprises one or morepharmaceutically acceptable carriers, diluents or excipients.Neutraceutical compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans; mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA; adjuvants and preservatives.

The neutraceutical may be an infant formula, particularly a humanisedmilk formula for administration to infants. Such an infant formula mayfind utility in treating failure to thrive or premature or low birthweight babies. It may also be administered to infants or children toimprove cognitive function.

The angiogenin used in the methods of the invention may be from anysource. It may be natural, synthetic or recombinant in origin.Recombinant angiogenin can be based on the angiogenin sequence from anyspecies, including humans, cows, sheep, mouse, etc. Recombinant humanangiogenin is available from R & D Systems.

Angiogenin is known to be present in normal human plasma, bovine plasma,bovine milk, bovine plasma and mouse, rabbit and pig sera. The DNA andprotein sequences of at least human angiogenin are available andrecombinant human angiogenin is available commercially from AbnovaCorporation (Taiwan) for small scale applications.

In one embodiment the angiogenin is prepared from plasma or milk fromlivestock animals as readily available sources of angiogenin on acommercial scale.

The milk may be obtained from any lactating animal, e.g. ruminants suchas cows, sheep, buffalos, goats, and deer, non-ruminants includingprimates such as a human, and monogastrics such as pigs. In a preferredembodiment the angiogenin is extracted from cow's milk. The animal fromwhich angiogenin is produced may be a transgeinic animal designed toover-express angiogenin in its milk.

The inventors of the present application have shown that in bovine milk,angiogenin is present in the highest or most concentrated amount (up to12 mg/litre) within the first 1 to 14 days of lactation. Following this,the concentration falls to a base level of approximately 1 to 2mg/litre. Therefore it is preferred that cow's milk which obtainedwithin the first 14 days of lactation as a source of angiogenin for usein the methods of the first to eleventh aspects. Given the residualangiogenin levels in cow's milk from later lactation, it may still beused a source for the methods of the invention.

The angiogenin used in the methods of the invention may be isolated orpurified. Purified or isolated angiogenin is substantially free of atleast one agent or compound with which it is naturally associated. Forinstance, an isolated protein is substantially free of at least somecellular material or contaminating protein from the cell or tissuesource from which it is derived. The phrase “substantially free ofcellular material” refers to preparations where the angiogenin is atleast 50 to 59% (w/w) pure, at least 60 to 69% (w/w) pure, at least 70to 79% (w/w) pure, at least 80-89% (w/w) pure, at least 90-95% pure, orat least 96%, 97%, 98%, 99% or 100% (w/w) pure.

Recombinant angiogenin preparations in bacteria may be used as a sourceof angiogenin and may be provided in the form of protein aggregates.

As bovine milk is a natural product that has been in food chain forhundreds of years, the angiogenin used as a nutraceutical need not betotally pure. However, to reduce the amount of composition to beadministered it is preferred that the angiogenin is concentratedsignificantly with respect to its concentration in milk. Preferably theangiogenin is administered in at a concentration of at least 10 timesits concentration in milk and more preferably 20, 30, 40, or 50 timesits concentration in milk.

When provided as a food the angiogenin can take the form of a foodsupplement, a nutritional formulation, a sports nutrition supplement oran infant formula.

Persons skilled in the art will appreciate that variants of bovineangiogenin exist in nature and can be manufactured. Use of such variantsis contemplated by the present invention.

One of skill in the art will recognize that angiogenin may contain anynumber of conservative changes its amino acid sequence without alteringits biological properties. Such conservative amino acid modificationsare based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary conservative substitutions which takevarious of the foregoing characteristics into consideration are wellknown to those of skill in the art and include arginine and lysine;glutamate and aspartate; serine and threonine; glutamine and asparagine;and valine, leucine, and isoleucine.

The present invention also includes the use of variants, homologues, andfragments of angiogenin. For example, the nucleic or amino acid sequencefor angiogenin may comprise a sequence at least 70% to 79% identical tothe nucleic or amino acid sequence of the native protein, or at least80% to 89% identical, or at least 90% to 95% identical, or at least 96%to 100% identical.

Persons skilled in the art would really appreciate the numerous softwarepackages to enable them to design or homologues of the angiogeninnucleotide and amino acid sequences, for example the “BLAST” program orother suitable packages.

It is understood by one of ordinary skill in the art that certain aminoacids may be substituted for other amino acids in a protein structurewithout adversely affecting the activity of angiogenin. It is thuscontemplated by the inventors that various changes may be made in theamino acid sequences of angiogenin without appreciable loss of theirbiological utility or activity. Such changes may include deletions,insertions, truncations, substitutions, fusions, shuffling of motifsequences, and the like.

In addition the angiogenin may be modified, for example byglycosylation, by conjugation to a polymer to increase their circulatinghalf-life, by pegylation or other chemical modification. Such modifiedproteins are also envisaged for use in the method of the presentinvention.

Persons skilled in the art will appreciate that the angiogenin used maybe modified to improve storage stability, bioactivity, circulating halflife, or for any other purpose using methods available in the art. Forexample it may be desirable to introduce modification to improve storagestability. However, as angiogenin is particularly resistant todegradation such modification may not be essential.

The invention refers to agonists of angiogenin. An agonist is a compoundthat is capable of directly or indirectly having an effect through thereceptor activated by angiogenin. Preferably angiogenin agonists actthrough the angiogenin receptor and preferably bind the receptor.Persons skilled in the art will appreciate how to design agonists ofangiogenin. Suitable agonists include angiogenin agonist antibodies andmimetic compounds.

Angiogenin, its agonists and variants may be used in the manufacture ofa medicament for use in the methods of the invention.

In a preferred embodiment of the methods and uses of the inventionangiogenin is administered orally, particularly in the form of anangiogenin enriched extract from milk or plasma or in the form ofrecombinant angiogenin

Particularly the orally administered angiogenin is prepared from cow'smilk or a fraction thereof, for example using the process described inexample 1. Such fraction has been found to provide angiogenin able toact systemically, without substantial degradation in the gut. Suchfraction is able to be provided orally without employing carriers orother mechanisms to enhance the bioavailability of angiogenin.

Angiogenin administered in accordance with the methods of the first toeleventh aspects is anticipated to interact with endogenous follistatin(if recombinant angiogenin is used) or the enriched angiogenin extractmay also contain follistatin. Administration of angiogenin plusfollistatin (either simultaneously or sequentially in any order) isshown herein to have a more than additive effect and accordingly acomposition comprising angiogenin and follistatin is provided, as wellas each of the methods of treatment contemplating administration offollistatin with angiogenin. It is particularly important toco-administer (either simultaneously or sequentially) follistatin withangiogenin in situations where an individual is follistatin deficient.As follistatin levels decrease with age, co-administration offollistatin with angiogenin is particularly contemplated when treatingthe elderly.

In a co-administration regime, angiogenin may be administered orally andfollistatin administered orally or otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

It must also be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

The invention is now further described in detail by reference to thefollowing example. The example is provided for purposes of illustrationonly, and is not intended to be limiting unless otherwise specified.Thus, the invention encompasses any and all variations which becomeevident as a result of the teaching provided herein.

Example 1 Process for the Preparation of an Angiogenin-Enriched Fractionfrom Skim Milk

A 10 cm deep column was packed with SP Sepharose Big Beads (GEHealthcare) such that the total bed volume of the column was 29.7litres. To the column a flow of skimmed cow's milk was applied at alinear flow rate of 331 cm/h (34 litres of skimmed milk per litre ofresin per hour) for 2 hours until the volume of skimmed milk applied was68 times the volume of the resin packed into the column.

The milk remaining in the column was removed by adding 2.5 columnvolumes (CV) of water at a linear flow rate of 147 cm/h (15 litres ofbuffer per litre of resin per hour), or 0.25 CV/min, for 10 min.

The angiogenin-depleted lactoperoxidase fraction was eluted from thecolumn with 2.5 CV of a buffer containing sodium ions equivalent to 2.0%(0.34M) NaCl, at pH 6.5, by flowing the cation buffer solution at alinear flow rate of 75 cm/h (7.5 litres of cation buffer solution perlitre of resin per hour), or 0.125 CV/min, for 20 min. The first 0.5litres of cation buffer solution per litre of resin was discarded todrain and the next 2.5 litres of cation buffer solution per litre ofresin was collected as the angiogenin-depleted lactoperoxidase fraction(including 0.5 litres of cation buffer solution per litre of resinoverlapping the application time of the next buffer, i.e. breakthroughtime).

The angiogenin-enriched fraction was then eluted from the column with2.5 CV of a buffer containing sodium ions equivalent to 2.5% w/v (0.43M)NaCl, at pH 6.5, by flowing the cation buffer solution at a linear flowrate of 75 cm/h (7.5 litres of cation buffer solution per litre of resinper hour), or 0.125 CV/min, for 20 min. The first 0.5 litres of cationbuffer solution per litre of resin was discarded to drain and the next2.5 litres of cation buffer solution per litre of resin was collected asthe angiogenin-enriched fraction (including 0.5 litres of cation buffersolution per litre of resin overlapping the application time of the nextbuffer).

Finally, the lactoferrin fraction was eluted from the column with 2.5 CVof a buffer containing sodium ions equivalent to 8.75% w/v (1.5M) NaCl,at pH 6.5, by flowing the cation buffer solution at a linear flow rateof 75 cm/h (7.5 litres of cation buffer solution per litre of resin perhour), or 0.125 CV/min, for 20 min. The first 0.5 litres of cationbuffer solution per litre of resin was discarded to drain and the next2.5 litres of cation buffer solution per litre of resin was collected asthe lactoferrin fraction.

The angiogenin-enriched fraction that was collected was ultrafiltrated(NMWCO 5 kDa) to concentrate and reduce the salt content. The resultantconcentrate was freeze-dried and stored at room temperature forsubsequent use.

The angiogenin-enriched fraction was analysed for angiogenin content bySDS-PAGE and the fraction was found to contain 57% (protein basis) of alow molecular weight (14 kDa) protein which was confirmed to beangiogenin by MALDI-TOF/TOF MS (results not shown).

Persons skilled in the art would appreciate that angiogenin from othersources or purified by other means could be used in the methods of theinvention. The above example is merely to show how the actual source ofangiogenin used in the following experiments was made and is in no wayintended to be limiting.

Whilst it may be considered that the angiogenin enriched fraction maycontain additional bioactive components which are having an effect, thecomparable amount of angiogenin as available in skim milk (concentration2%) had comparable activity in the examples shown to the angiogeninenriched fraction (data not shown).

Example 2 In Vitro Analysis—Bovine Angiogenin is Active on Human Cells

Angiogenin was provided in an enriched extract prepared from bovineskim-milk according to the method described above.

An angiogenesis assay employing human umbilical vein endothelial cells(HUVECS) was used to determine if bovine angiogenin is active on humancells. HUVEC cells were routinely maintained in Endothelial cell basal(ECB) medium, supplemented with bovine brain extract, EGF,hydrocortisone and 10% FBS (Clonetics). Assays were performed intriplicate in 48 well tissue culture plates. 150 μl of Matrigel (BDbiosciences) was first allowed to polymerise on the bottom of each well.HUVEC cells were resuspended in ECB with now 1% FBS and bovineangiogenin, at 0.5×10⁶ cells/ml. The cells (2.5×10⁴ cells/well) werethen plated on to the matrigel matrix and incubated at 37° C. for 24hours. Human vascular endothelia growth factor (VEGF) 10 ng/ml replacedangiogenin as a positive control and ECB media 1% FBS alone was used anegative control. Vascular development was observed and photographed at10× magnification and the results shown in FIG. 1.

The results show that bovine angiogenin induces vascular development ofHUVEC on matrigel in the same manor as human VEGF and therefore bovineangiogenin is shown to be active on human cells.

Example 3 In Vitro Muscle Cell Growth Assays

Muscle cells (C2C12; mouse myoblasts) were seeded into 96-well plates ata starting density of 1×10⁴ cells/well in Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal bovine serum (FBS). The cells werecultured overnight at 37° C.; 5% CO₂. The next day the serum containingmedia was removed and the cells washed in PBS. The cells were thencultured in 100 μl serum free DMEM supplemented with test agents (n=7)for 48 hrs at 37° C.; 5% CO₂. To quantitate the cell growth 10 μl WST-1cell proliferation reagent (Roche) was added to each to each well andthe cells incubated for a further 3 hours at 37° C. During this timeviable cells convert the WST-1 reagent to a soluble formazan dye whichwas measured in a microplate reader, the absorbance at 450 nm directlycorrelates to the cell number. Stimulation of cell growth by the agentswas compared to a positive control (10% FCS) and negative controls(DMEM+vehicle control; DMEM containing BSA or casein at appropriateprotein loads).

For muscle cell differentiation studies, C2C12 myoblasts were seededinto the 6 well plates at 25×10⁴ cells in 2 ml of media (DMEM, 10% FBS)and allowed to attach overnight. To induce differentiation intomyotubes, the culture media was removed and replaced with DMEM alone orDMEM supplemented with 2% horse serum. The effects of bAngiogenin (0.1μg/ml-100 μg/ml; 10 μg/ml where not stated), rhAngiogenin (0.1 μg/ml-10μg/ml; 1 μg/ml where not stated), rhFollistatin (0.1 μg/ml) andrhMyostatin (50 ng/ml) were tested. All recombinant proteins werepurchased from RnD Systems. Images of cells were taken and creatinekinase (CK) activity was measured after treatment for 96 hours, or 48hours for experiments involving myostatin. We measured Creatine Kinaseactivity activated by N-Acetyl Cysteine (NAC) according to themanufacture's instructions. Briefly for each assay a fresh vial ofCK-NAC reagent (Thermo Cat #TR14010) with made up with 10 ml of sterilewater. 17.5 μl of each sample was then mixed with 350 μl CK-NAC reagentand triplicate 100 μl aliquots assayed in 96 well plates. The absorbanceat 340 nm was then measured for five minutes. CK activity was calculatedfrom the change in abs/min using the following equations:

${{Activity}\mspace{14mu} ( {U/L} )} = {{{\Delta \; {abs}\mspace{14mu} \min} - {1 \times {Factor}\begin{matrix}{{Factor} = \frac{{Total}\mspace{14mu} {vol} \times 1000}{6.3 \times {sample}\mspace{14mu} {vol} \times {cuvette}\mspace{14mu} {pathlength}}} \\{= {0.1 \times {1000/( {6.3 \times {.005} \times 1} )}}} \\{= 3174.6}\end{matrix}{CK}\mspace{14mu} {Activity}\mspace{14mu} ( {U/L} )}} = {{\Delta \; {abs}\mspace{14mu} \min} - {1 \times 3174.6}}}$

For microarray analyses of cell cultures, total RNA was extracted fromcultured cells using the RNeasy mini RNA isolation kit (Qiagen) andquantitated by measuring absorbance at 260 nm with the Nanodrop 1000spectrophotometer. Purity was also assessed by obtaining 260 nm/280 nmand 260 nm/230 nm ratios. RNA integrity was assessed by running a sampleof each RNA on the Bioanalyser 2100 using the RNA 6000 Nano LabChip kit(Agilent).

100 ng of total RNA was amplified to produce biotin-labeled cDNA usingthe GeneChip® Whole Transcript (WT) Sense Target Labeling Assay(Affymetrix) as per the protocol provided by the manufacturer. LabelledcDNA was applied in recommended quantities to Mouse Gene 1.0 ST Arrays(Affymetrix) before being washed and stained using the Affymetrix 450Fluidics Station and recommended solutions (Affymetrix). Scanning of thearrays was done on the Affymetrix GeneChip® Scanner 3000 7G beforeintensity data was extracted using Affymetrix GeneChip® Command Console(AGCC) software. The resultant .CEL files were used for data analysis inPartek® Genomics Suite ver. 6.4 (Partek), using default RMAnormalisation and ANOVA

The results of muscle cell differentiation experiments are shown inFIGS. 3, 4 and 5. FIG. 3 shows that under serum free non-differentiationconditions, bovine and rh angiogenin allow muscle cell differentiationto form myotubes. FIG. 4 shows that bAngiogenin also increases myoblastdifferentiation and myotube formation in the presence of 2% HS in a dosedependant manner. Inclusion of rhAngiogenin at a single dosage levelproves that angiogenin is the inducing factor. FIG. 5 shows thesynergistic effects of bovine angiogenin and rh follistatin. Undernormal differentiation conditions, myotube size is increased followingculture with bAngiogenin and rhFollistatin compared to standardconditions or culture with angiogenin or follistatin in isolation (FIG.5 a). Increased differentiation is proved by creatine kinase assays (5b) showing significantly higher levels in the angiogenin andrhFollistatin combination treatment compared to the treatments inisolation or the control.

The synergistic effects of bAngiogenin and rhFollistatin on global geneexpression profiles during the initial phase of myotubes formation(first 2 hours following differentiation) were tested using microarrayanalysis (FIG. 6). Minor differences in gene expression profiles areobserved during initial myoblast differentiation in the controltreatment or in presence of rhFollistatin or bAngiogenin. Markeddifferences are seen in the rhFollistatin and bAngiogenin combinationtreatment compared to the other treatments.

The specific role of angiogenin in the differentiation process wastested by repeating the differentiation culture conditions with thepeptide VFSVRVSILVF (AUSPEP) which specifically blocks theangiogenin/actin interaction (FIG. 7). The angiogenin blocking peptideinhibited bAngiogenin specific differentiation as measured by increasedcreatine kinase activity compared to the control group, demonstratingthat the response observed in FIG. 5 is due specifically to angiogenin.

The ability of angiogenin to recover muscle cell differentiation wastested by incubating C2C12 muscle myoblasts with rhMyostatin,bAngiogenin and rhFollistatin under differentiation conditions.Myostatin is a negative regulator of muscle cell differentiation andinhibits binds with high affinity to follistatin. FIG. 8 demonstratesthat myostatin inhibits muscle cell differentiation to myotubes and thatfollistatin alone can not recover cell differentiation. IncludingbAngiogenin in the incubation media recovered the majority of creatinekinase activity, however, the combination of bAngiogenin plusrhFollistatin recovered creatine kinase levels to the control levels,showing that angiogenin circumvents normal myostatin-follistatin celldifferentiation signalling. This experiment was repeated usingrhAngiogenin to prove the specificity of the mechanism to angiogenin(FIG. 9). This shows that using rhAngiogenin, the recovery of myostatininduced reduction in creatine kinase activity is identical to that ofbAngiogenin, including the synergistic mechanism with follistatin.

Example 4 Angiogenin is Neuroprotective

To test if angiogenin was active in combination with follistatin onnerve cells PC12 cells were cultured with bAngiogenin, rhAngiogenin, andrhAngiogenin+rhFollistatin and cell survival measured in the absence ofserum. FIG. 10 and FIG. 11 show protection of PC12 cells against celldeath upon serum starvation in the presence ofrhAngiogenin+rhFollistatin relative to rhAngiogenin alone orrhFollistatin alone. Bovine angiogenin also had a protective effect.After 22-24 hr of pre-treatment in the presence of treatments incomplete medium (in DMEM with 10% horse serum and 5% heat-inactivatedFBS), the cells were washed twice with 300 μl/well of serum free DMEMand addition of protein reagents. After three days of incubation, cellviability was measured based on ATP levels using CellTiterGlo® reagent(Promega, Madison, Wis.). Luminescence was read using a Victor3 (PerkinElmer, Waltham, Mass.) multilabel plate reader at room temperature. FIG.10 and FIG. 11 show protection of PC12 cells against cell death uponserum starvation.

Example 5 In Vivo Animal Studies

To analyse the in vivo effects of angiogenin on muscle phenotype innormal and muscular dystrophic mice animal studies were undertaken. Allwork was approved by the University of Western Australia animal ethicscommittee.

Mice were fed 2 diets during each trial; a control diet and a dietcontaining an bAngiogenin enriched fraction made according to example 1at 2.5 μg/g mouse weight. These studies were carried out on adult (8 wksof age) male normal (C57) and dystrophic (mdx) mice with n=8 for eachmouse strain per diet for each experiment.

Normal mice were subjected to a one month dietary period with ad libitumaccess to feed and voluntary exercise; for voluntary exercise a metalmouse wheel is placed inside the cage and the distance run by individualmice is recorded by a bicycle pedometer attached to the wheel. MDX micewere subjected to the same one month dietary period. In separateexperiments, mdx mice were given the voluntary exercise treatmentdescribed above or were given no voluntary exercise wheel.

Experimental Analysis:

During the experiments body weight, amount of food eaten and musclestrength (grip strength test) were all measured twice weekly. At theconclusion of each experiment the mice were sacrificed by halothaneanaesthesia and cervical dislocation.

Experimental mice were used for the following analysis to determine anychanges in phenotype as a result of treatments on dystrophic and normalmuscle.

1) Body Composition analysis: Half of each skinned mouse carcase wereanalysed for body composition. In addition, individual leg musclesincluding the quadriceps (quad), tibialis anterior (TA) andgastrocnemius muscles were dissected, and weighed, as well as theabdominal fat pads and heart, data was recorded to determine grossphenotypic changes induced by the diets.

2) Histological analysis: Skeletal muscle and heart samples werecollected and prepared for both frozen and paraffin histology.Histological analysis was performed on the following muscles, quad, TAand diaphragm. Haematoxylin and Eosin, Sudan Black and variousimmunohistological stains were performed on these muscles. Skeletalmyofibre necrosis, myofibre hypertrophy and fat content of muscles wasdetermined.

Results from the in vivo experiment are shown in FIGS. 12 to 15. It isclear that the diet supplemented with bAngiogenin enriched fraction at2.5 μg/g induces muscle gain (FIG. 12) of up to 50% compared to thecontrol group. Increase in muscle mass was accommodated by increasedcross sectional area of most muscle fibre types except for thepopulation of small dark fibres corresponding to slow-twitch oxidativefibres (FIG. 13). Mice receiving the angiogenin enriched diet also ran30% further than the control diet mice as measured by voluntary exercise(FIG. 14). Taken together, this data shows that angiogenin influencesmuscle size and fitness in vivo.

When fed to mdx mice, angiogenin reduced the proportion of the musclethat was necrotic when mice were allowed access to voluntary exercise(FIG. 15). This demonstrates that angiogenin is capable of inhibitingthe effects of exercise on muscle breakdown in mdx mice.

1-22. (canceled)
 23. A method of treating a disorder characterised byelevated or dysregulated myostatin in an individual, treating disorderswhere the interaction between follistatin and angiogenin can be used toimprove function in tissues, promoting muscle growth in an individual,improving recovery of muscle from injury or use in an individual,improving muscle strength in an individual, improving exercise tolerancein an individual, increasing the proportion of muscle in an individual,decreasing fat in an individual, treating neurological diseases ordisorders, spinal injuries or diseases, bone diseases or disorders,diseases involving glucose homeostasis, wound healing, or for providingneuroprotection, nervous system functional support, managing metabolicdiseases; or decreasing an individual's fat to muscle ratio, the methodcomprising administering an effective amount of angiogenin or anangiogenin agonist.
 24. The method of claim 23, wherein the disorder isselected from the group consisting of metabolic diseases,insulin-dependent (type 1) diabetes mellitus, noninsulin-dependent (type2) diabetes mellitus, hyperglycemia, impaired glucose tolerance,metabolic syndrome, syndrome X, insulin resistance induced by trauma,adipose tissue disorders, obesity, muscle and neuromuscular disorders,muscular dystrophy, severe or benign X-linked muscular dystrophy,limb-girdle dystrophy, facioscapulohumeral dystrophy, myotinicdystrophy, distal muscular dystrophy, progressive dystrophicophthalmoplegia, oculopharyngeal dystrophy, Duchenne's musculardystrophy, and Fakuyama-type congenital muscular dystophy; amyotrophiclateral sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpaltunnel syndrome; congestive obstructive pulmonary disease; congenitalmyopathy; myotonia congenital; familial periodic paralysis; paroxysmalmyoglobinuria; myasthenia gravis; Eaton-Lambert syndrome; secondarymyasthenia; denervation atrophy; paroxymal muscle atrophy; andsarcopenia, cachexia, other muscle wasting syndromes, osteoporosis,especially in the elderly and/or postmenopausal women;glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis;osteoporosis-related fractures; traumatic or chronic injury to muscletissue, low bone mass due to chronic glucocorticoid therapy, prematuregonadal failure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa. 25.The method of claim 23, wherein the method treats metabolic syndrome orenhances insulin sensitivity.
 26. The method of claim 23, wherein theangiogenin is bovine in origin.
 27. The method of claim 26, wherein theangiogenin is extracted from bovine milk.
 28. The method of claim 23,wherein the angiogenin is administered orally without carriers ormodification being required to allow for oral bioavailability.
 29. Themethod of claim 23, wherein angiogenin or an angiogenin agonist isadministered with follistatin (either simultaneously or sequentially).30. The method of claim 23, further comprising administering aneffective amount of follistatin.
 31. A composition, food supplement, orneutraceutical comprising angiogenin or an angiogenin agonist in aformulation suitable for treating a disorder characterised by elevatedmyostatin, for treating disorders where the interaction betweenfollistatin and angiogenin can be used to improve function in tissues,for promoting muscle growth, for improving recovery of muscle frominjury or use, for improving muscle strength, for improving exercisetolerance, for increasing the proportion of muscle, for decreasing fat,for decreasing an individual's fat to muscle ratio, for treatingneurological diseases or disorders, for treating spinal injuries ordiseases, for treating bone diseases or disorders, for treating diseasesinvolving glucose homeostasis, for wound healing, or for providingneuroprotection, nervous system functional support, managing metabolicdiseases and/or increasing the bone density of an individual.
 32. Thecomposition of claim 31, further comprising follistatin.
 33. A methodfor improving the bone density of an individual by administering aneffective amount of angiogenin or an angiogenin agonist in combinationwith follistatin.